This invention is in the field of pharmacology, and relates specifically to the pharmacological treatment of conditions associated with herpes simplex virus infections.
2. Description of the Prior Art
Herpes Simplex--the Virus
No human virus is considered normal flora; although some viruses may be more or less symptomatic, unlike bacteria none can be considered non-pathogenic. And because the viral life cycle is played out within a host cell, the membrane and molecular function of the target eukaryocyte and the biological life cycle of the invasive virion are inextricably entwined.
Viruses may be grouped in a variety of ways; perhaps most simply by considering five elements:
1) Method of entry into the host. PA1 2) Extent of spread in the host. PA1 3) Mode of spread within the host. PA1 4) The host tissue targeted. PA1 5) The fate of the virus after host recovery. PA1 1) the great variability of the relative amounts of lysine and arginine in diets; and PA1 2) failure to measure the serum lysine concentration. (The latter should be maintained above 165 nmol/mL) PA1 1. Retarding infected host cell clumping by reducing surface glycoprotein. This reduction of cell clumping mechanically interferes with virion-host cell fusion and interferes with cell-to-cell spread. (2-deoxy-D-glucose) PA1 2. Retarding virion-host cell fusion physiologically by reducing levels of glycoprotein gB carbohydrate and, thus, reducing virus infectivity. (2-deoxy-D-glucose) PA1 3. Modifying the host-cell immunomodulation abilities and thus improving existing statistical differences in herpes recurrence rates between patients with dietary L-arginine/L-lysine imbalances. (L-lysine) PA1 4. Improving host-cell defenses by increasing endogenous reduced-GSH levels. Maintenance of GSH levels interferes with late-stage replication of HSV-1. (Se.sup.2+, NAC) PA1 5. Improving local host-cell antiviral antioxidative effectiveness by reducing local levels of hydroxyl radicals involved in the pathogenesis of ophthalmic herpes. This improves local tissue survival by countering high levels of free radical damage. (Se .sup.2+) PA1 6. Increasing host-cell levels of antioxidant thiols and glutathione peroxidase and, in addition, providing synergistic anti-replicative activity in conjunction with acyclovir and deoxyuridine. (quercetin, NAC) PA1 7. Inactivating viral replication and reducing host-cell levels of hydroxyl radicals. (Cu.sup.2+, ascorbate--alone or, more effectively, in combination) PA1 8. Locally inactivating HSV without creating long-term resistance and concurrently ensuring adequate immune system stability. (Zn.sup.2+) PA1 9. Inhibiting primary virus fusion by blocking access to the heparan sulfate receptor and improving lesion healing by stimulating bFGF mediated reparative angiogenesis. (heparin sodium) PA1 10. Inhibiting viral DNA synthesis and thus reducing viral replication. (2-deoxy-D-glucose)
According to this admittedly simple list of characteristics, herpes simplex virus (HSV), Herpesviridae, Simplexvirus, enters the host by direct contact, is spread to a target tissue only, spreads within the host via neuronal axonal flow, targets the dorsal root ganglia and after recovery of the host from an acute infection, remains latent in the targeted tissue.
The HSV virion is a large (100 to 150 m.gamma.), enveloped virus with an icosahedral capsid. It has double strand DNA with a genome that encodes at least 70 polypeptides--this large amount of regulatory information permits the virus to control its own gene expression and elegantly to modify multiple complex events within the infected cell.
The invading virion binds to host cell receptors. A primary binding site is host cell surface heparan sulfate glycosaminoglycan, which binds with the V3 loop of the viral envelope glycoprotein (gp 120). Another primary binding site may be chondroitin sulfate. Mediated by viral glycoprotein gB and following nonspecific primary binding, more specific binding occurs to the gC4 and gD4 viral surface glycoproteins. The virion envelope fuses with the plasma membrane of the host cell. The capsid is uncoated, the virus invasively inserts surface glycoprotein gB through the host cell plasma membrane and enters the host nucleus where viral DNA is transcribed and processed into mature mRNA; at the same time, host cell mRNA synthesis is inhibited. Invading HSV also inhibits host cell DNA synthesis while viral DNA replicates within the host nucleus. The viral DNA combines with newly formed HSV capsid proteins translated in the cytoplasm, and assembles into progeny virion particles within the nuclear membrane. Concurrent expression of glycoproteins in the host plasma envelope stimulates neighboring cells to clump together. Following cell-to-cell contact by binding and fusion of their respective plasma envelopes, progeny particles invade clumped, neighboring host cells directly or by spread following lysis of previously invaded tissue cells or phagocytes and the process repeats itself.
Viral invasion elicits a phagocytic response coupled with typical phagocytic immune activities--the release of soluble immune mediators (i.e., cytokines) and high respiratory burst responses by activated phagocytes. These immune responses are themselves detrimental to the host; not only because of local tissue necrosis from high environmental levels of free radical release, but also because of the development of mutant, potentially resistant viral strains secondary to toxic local levels of activated oxygen and hydroxyl species.
Herpes Simplex--Clinical Expression
The massive disruption by HSV of host cell molecular functions and of host cellular structure is manifested clinically as host cellular death, resulting in shallow, painful vesicular ectodermal lesions or by hemorrhagic encephalitic necrosis of the brain. Target tissues for HSV are the skin or mucous membranes usually derived from embryonic ectoderm: mouth, skin, vagina, conjunctiva, cornea, etc. The virus enters the host cell by direct mucosal contact or by direct contact of abraded skin. In the skin the virus replicates in epithelial cells and then enters local sensory neurons. The virus travels to the dorsal root ganglia via retrograde axonal flow where it establishes permanent residency. There it establishes latency a state in which the viral lytic genes are silenced and only the latency locus is transcriptionally active. Although latent most of the time, it reactivates intermittently, travels down the sensory nerve and causes vesicular eruptions at or near the site of initial invasion. Alternatively the virus may invade the CNS and cause encephalitis.
The rate of seropositivity to HSV varies widely from country to country:
from relatively low in Japan where Herpes simplex Type 1 (HSV-1) seroprevalence for men and women has decreased from 75.3 and 80.6% in 1973 to 54.4 and 59.6%, respectively in 1993 and where Herpes simplex Type 2 (HSV-2) seroprevalence has decreased from 10.2 and 9.9% in 1973 to 1.8 and 1.2%, respectively in 1993, to quite high in Africa where all adult study groups have a high HSV-1 seroprevalence of &gt;80%.
HSV infects more than 50% of the adult population, but some infections may be unrecognized. About half of these develop clinical manifestations of the disease. Its most significant manifestations are keratitis, genital lesions and labial vesicular lesions ("cold sores").
HSV-1 typically causes herpes keratitis (cornea). This disease is identified by a typically bizarre dendritic-patterned corneal ulcer that tends to be recurrent and very often leads to scarring with a reduction of vision, sometimes to the level of legal blindness. HSV-1 also causes herpes labialis, peri-orbital, peri-oral, peri-nasal skin eruptions and, in older patients, the virus has been associated with herpes zoster ("shingles") infection of the upper trunk.
HSV-2 causes the most prevalent sexually transmitted disease in the United States and visits to physicians for genital herpes simplex virus infection continue to increase. As many as 30 million Americans are infected with HSV-2. About half of these carriers are symptomatic. The clinical manifestations range from mild genital inflammation to severe, very painful, vesicular lesions and ulceration. Systemic involvement in the most severe cases may include hepatitis. Brain damage and death often are the result of HSV-2 acquired by a newborn infant as it passes through an infected birth canal.
Once the herpes virus (of either kind) has infected the human body, the virus is permanently present. This is particularly true for viral infection of the nerve cells of the dorsal root ganglia that are out of range of the immune system. Less commonly, the epithelial basement membrane may house the latent virus. The virus becomes periodically active when the immune system is depressed or when oxidative stress is increased, i.e., during illness, after exposure to high intensity ultraviolet light, following local tissue trauma, etc.
Although HSV-1 principally causes corneal infections or "cold sores" and HSV-2 most often causes genital herpes, either type can infect the cornea, the mouth and/or the genitals. Similarly although most herpetic ocular infections in adults are caused by HSV-1, other more severe and prolonged cases in adults have been shown to be caused by HSV-2.
Herpes Simplex--Current Clinical Treatment
Present treatment rationales are focused upon preventing the fusion of the virion envelope with the host cell plasma membrane by negatively influencing host cell membrane receptors or by interfering with the glycosylation of viral protein required for fusion, and by reducing viral replication within the host cell nucleus. More recently some attention has been drawn to the relationship between local levels of toxic free radicals and antioxidants in the host target cell environment and apparent target cell resistance to infection following viral reactivation.
A. Ophthalmic Preparations:
1. .alpha.-.alpha.-.alpha.-trifluorothymidine--(Viroptic.RTM. 1% solution)--useful in treating HSV-1 and HSV-2 keratoconjunctivitis, i.e., HSV lesions of the conjunctival and corneal epithelium, but not effective in the treatment of associated corneal stromal lesions. It acts by interfering with thymidine synthesis in eukaryocytes, normal or infected. Its precise action against invading viruses is unknown. Little clinical toxicity is described, but pregnant women should use it with caution. 2. 2'-Deoxy-5-idouridine--(Herplex.RTM. 0.1% solution)--useful in the treatment of corneal epithelial infection with HSV-1. The delivered solution is converted to idoxuridine which replaces DNA thymidine involved in the enzymatic step of viral replication. The resulting structural faults in viral DNA prevent replicative tissue infection. However, idoxuridine is generally cytotoxic, crosses the placental barrier and is implicated in fetal malformations in rabbits and rats. Pregnant women should use it with caution.
3. 9-.beta.-D-arabinofuranosyladenine--(Vira-A.RTM. 3% ointment--useful in the treatment of corneal epithelial HSV-1 and HSV-2 infections, but not stromal lesions induced by these viruses. Although the mode of action of Vira-A.RTM. is not established, it probably acts by interference with viral DNA synthesis. Embryonic mutogenesis has occurred in male germ cells and mouse embryos.
B. Genital Herpes Preparations:
1. acyclovir--(Zovirax.RTM. tablets)--useful in the treatment of HSV-1 and HSV-2 as well as other virus infections. Mode of action appears to be interference with viral DNA polymerase resulting in premature termination of the DNA chain and a reduction of viral replication. May be effective in preventing corneal stromal infection if used prophylactically, but expense (A major pharmaceutical wholesale firm, Henry Schein, list prices which range from $2.34 to $4.58 per tablet.), concerns for general cytotoxity and especially the rapid, irreversible development of resistant viral strains, limits this routine use. The use of acyclovir results in the emergence of highly resistant viruses sometimes with only one pass of therapy. Low rates of teratogenicity have been found in rats exposed to acyclovir.
Herpes Simplex--Antiviral Agents Under Study
1. 2-deoxy-D-glucose (glucosamine)--Glycosylation inhibitors such as 2-deoxy-D-glucose have been shown to retard the appearance and speed the evolution of both HSV-1 and HSV-2. There are several steps in the metabolism of virus-induced cellular surface glycoproteins that induce infected cell clumping that may be negatively affected by glucosamine. In similar fashion, by inhibiting glycosylation and thereby reducing levels of surface glycoprotein gD and gB, glucosamine reduces virion-host cell fusion; fusion is inhibited in the presence of reduced levels of viral surface glycoprotein carbohydrate. 2-Deoxy-D-glucose has also been shown to inhibit viral DNA synthesis (human cytomegalovirus) thus reducing viral replication potentials.
2. L-lysine monohydrochloride--Topical application of L-lysine to the skin of guinea pigs protected the skin from HSV inoculation. It is suggested that LMH exerts an immuno-modulatory effect in the herpes simplex host. More specifically, a study involving 52 subjects indicates that oral LMH is an effective agent for the reduction of occurrence, severity and healing time for herpes simplex virus infections. One study reported that subjective improvement seemed to occur in 88% of herpes simplex patients using L-lysine. However, there are studies in which L-lysine is reported to be ineffective with daily dosages below about 1000 milligrams per day. At least one study found that L-lysine had no effect on the rate of healing or the appearance of lesions. The conflicting results obtained for the efficacy of lysine for herpes infections may be explained by:
The higher the arginine/lysine ratio in any diet, the greater the risk for herpes recurrence. Patients with diets high in naturally occurring arginine, such as legumes, whole grains, and nuts, are more vulnerable to herpes simplex recurrence than those whose diets are high in lysine, such as meat and dairy products. The mean daily intakes of lysine and arginine for 16 persons studied were 8.11g.+-.2.28 and 6.32g.+-.1.74, respectively. The standard deviations from the mean intake levels are notably wide and most likely illustrate the large variability of lysine and arginine intake in individual diets. This widely variable dietary intake underlines the value of dietary supplementation in countering herpes simplex virus infections; a better dietary balance between these two amino acids should help reduce the existing statistical difference in herpes recurrence.
3. Glutathione (GSH) and Selenium (Se.sup.2+)--In vitro studies show that intracellular, endogenous, reduced GSH levels are significantly and immediately decreased in the first 24 hours after herpes virus invasion. This dramatic cellular depletion emphasizes the importance of GSH in the host cell's defense against the virus. Supplementation with exogenous GSH not only restored intracellular levels almost to those found in uninfected cells, but also inhibited over 99% of the replication of HSV-1. Although, GSH interferes with the late replication stages of the HSV-1 cycle, it does not disturb normal cellular metabolism.
Human GSH levels cannot be raised directly by supplemental administration in the diet. GSH is produced intracellularly from precursor amino acids including glycine and cysteine. One GSH precursor, N-acetyl-L-cysteine (NAC)--a high endogenous thiol in redox status--has itself been found to possess antiviral antioxidative effectiveness. This study suggested that a high thiol redox status may contribute to the apparent barrier function of endothelial cells with respect to viral infection (in this case, cytomegalovirus) and that oxidative stress may facilitate infection of the vascular wall. In fact, the activity of antioxidants such as glutathione reductase, glutathione peroxidase and Cu--Zn superoxide dismutase appear to be reduced in the lacrimal fluid of patients with herpes simplex keratitis and are altered during the active phase of the disease. Impaired inhibition of the hydroxyl radical and a drop of antioxidant activities in herpes-infected cornea and tears appear to be factors in the pathogenesis of ophthalmic herpes. The trace element Zn.sup.2+, plays an important, if indirect, role here because it function as a cofactor for the Se.sup.2+ -dependent protective enzyme glutathione peroxidase.
4. Quercetin--In an in vitro cell culture study, the naturally occurring flavanol 3,3 ',4',5,7-pentahydroxyflavone (quercetin) caused a concentration-dependent reduction of infectivity of a number of viruses, including HSV-1. In addition, it reduced intracellular viral replication. This activity may be related to the ability of quercetin to increase non-protein -SH compounds (important anti-oxidant agents) and increase glutathione peroxidase activity. Yield reduction studies (chick embryo fibroblasts) reveal that quercetin acts synergistically with acyclovir and with 5-ethyl-2'-deoxyuridine to enhance the HSV-1 and HSV-2 antiviral activity of these widely used clinically pharmaceuticals.
5. Ascorbate, ascorbic acid and Copper (Cu.sup.2+)--Impaired inhibition of hydroxyl radicals and reduced levels of ascorbic acid in the corneae and tears of herpes-infected eyes are factors in the pathogenesis of ophthalmic herpes. Suspensions of HSV have been inactivated by copper-catalyzed sodium ascorbate. Although inactivation of herpes simplex virus can be achieved by Cu.sup.2+ used alone, this effect is enhanced by the addition of ascorbate. One study mentions that a topical paste consisting solely of vitamin C was effective in the treatment of HSV lesions.
6. Zinc (Zn.sup.2+)--Zinc sulfate inactivates free herpes simplex virus. Zn.sup.2+ inactivation of the virus lessens after several passes, but this partial resistance of the virus eventually disappears. (In contrast, resistance to acyclovir is complete and irreversible after a single pass.) Consistent with this in vitro evidence of the persistence of zinc's inhibitory effect on HSV, is the finding that long-term, topical application of Zn.sup.2+ greatly reduces or eliminates recurrences of genital herpes. Even low concentrations of zinc, prevented recurrent herpes simplex. These direct contact effects of zinc on HSV reflect and complement the systemic importance of Zn.sup.2+ in global immune system maintenance. For example: in rabbits Zn.sup.2+ plays a vital role in maintaining immunocompetence. Humoral and cellular immunity are depressed in the Zn.sup.2+ -deficient rabbit. Epithelial and stromal HSV keratitis are more severe in the Zn.sup.2+ -deficient rabbit and these conditions are not improved by local Zn.sup.2+ replacement used alone (zinc sulfate ointment). This treatment failure highlights the necessity of maintaining a healthy underlying immune system in resisting HSV and the important involvement of dietary Zn.sup.2+ in maintaining that immunity.
7. Magnesium (Mg.sup.+2)--The recommended daily allowance of ionic Mg.sup.+2 for humans is 350 mg. Mg.sup.+2 deficiencies have been documented in many segments of the world population. It is estimated that the average adult in Western society has a dietary magnesium shortfall of 90-178 mg per day. Mg.sup.+2 deficiencies are particularly prevalent among diabetics with normal renal function, alcoholics, smokers, the elderly, and those who suffer from a variety of gastrointestinal mobility disorders.
Ionic Mg.sup.+2 in mammals resides in three compartments: (1) in bone; (2) in an intracellular bound form or in an intracellular unbound form; and (3) in circulating bound and unbound forms. When the concentration of circulating Mg.sup.+2 in the bloodstream increases as a result of the dietary uptake of Mg+.sup.2, the body quickly responds by sequestering the Mg.sup.+2 into one of the bound or intracellular forms listed above. If elemental Mg.sup.+2 is ingested in a bulk amount that results in the absorption of a Mg.sup.+2 bolus in excess of 8 mEq, the renal excretion of Mg.sup.+2 rapidly increases and, as a result, becomes less efficient in the resorption of this element. Thus the accurate sustenance of an appropriate Mg.sup.+2 level requires the repeated administration of carefully designed Mg +2-containing medicaments with correctly formulated, absorption targeted amounts.
Among other functions, Mg.sup.+2 and Cu+.sup.2 deficiencies impair antioxidant defenses through decreased synthesis of GSH and reduced activity of CuZn superoxide dismutase, respectively. Mg.sup.+2 deficiencies enhance general oxidative stress levels by permitting elevated circulating levels of factors that promote free radical generation and which are mitogenic. This results in increased tissue necrosis in the presence of acute local levels of active oxygen species or hydroxyl radicals.
7. Heparin Sodium--Heparan sulfate is a primary receptor for viral fusion with the host cell. Very low doses of sodium heparin bind competitively with host cell surface heparan sulfate receptors and thus inhibit the very earliest stages of virion fusion. In addition, heparin sodium mobilizes fibroblastic growth factor (bFGF) by releasing it from its bound status to heparan sulfate. bFGF is a potent mediator of inflammatory angiogenesis fundamental to lesion repair. The effective doses of heparin sodium required for these activities are greatly lower than those necessary for anticoagulant purposes.